Inorganic pyrophosphatase (pyrophosphate phosphohydrolase) (PPase) is an enzyme that plays an important role in energy metabolism. This enzyme is responsible for the hydrolysis of pyrophosphate (PP.sub.i) which is formed principally as the product of the many biosynthetic reactions that utilize nucleoside triphosphates, such as DNA and RNA synthesis, coenzyme synthesis and the activation of amino acids and fatty acids. This enzyme is considered to maintain the forward direction of these reactions and is vital for maintaining viability. For a review of microbial inorganic pyrophosphatases, see, Lahti, R., Microbiol. Review (1983) 47:169-179, the disclosure of which is hereby incorporated by reference herein.
Inorganic pyrophosphatases occur widely in nature. The two best-studied PPases are those from Saccharomyces cerevisiae and Escherichia coli. See, for example, Cooperman, B. S., Methods Enzymol. (1982) 87:526-548, and Josse, J. and Wong, D. C. K. in Enzymes (1971), 3rd Ed., Vol. 4 (Boyer, P. D., ed.) pp. 499-527, Academic Press, New York. Research (the disclosure of which is hereby incorporated by reference herein), has also been conducted on the isolation and purification of inorganic pyrophosphatases from other microorganisms. Four PPases have been isolated from the Archaea: Methanobacteriun thermoautotrophicum (strain .DELTA.H) in Alebeek, et al., Biochim. Biophys. Acta (1994) 1206:231-239, the disclosure of which is hereby incorporated by reference herein; Methanothrix soehngenii in Jetten, et al., Arch. Microbiol. (1992) 157:284-289, the disclosure of which is hereby incorporated by reference herein; Thermoplasma acidophilum in Richter and Schafer, Eur. J. Biochem. (1992) 209:343-349, the disclosure of which is hereby incorporated by reference herein; and Sulfolbus acidocaldarius strain 7 in Wakagi, et al., Biochim. Biophys. Acta (1992) 1120:289-296, the disclosure of which is hereby incorporated by reference herein, and S. acidocaldarius (DSM 639) in Meyer, et al., Arch. Biochem. Biophys. (1995) 319:149-156, the disclosure of which is hereby incorporated by reference herein. Furthermore, the 3-dimensional structure of three PPases has been determined by X-ray crystallography: Saccharomyces cerevisiae (Baker's yeast) in Arutiunian, et al., Dokl. Akad. Nauk. SSSR (1981) 258:1481, the disclosure of which is hereby incorporated by reference herein; E. coli in Kankare, et al., Protein Engineering (1994) 7:823-830, the disclosure of which is hereby incorporated by reference herein; and Thermus thermophilus in Teplyokov, et al., Protein Science (1994) 3:1098-1107, the disclosure of which is hereby incorporated by reference herein.
Two different categories of inorganic pyrophosphatases have been differentiated: soluble (cytoplasmic) and membrane-bound. This differentiation is based not only on cellular localization, but on subunit structure. Cytoplasmic PPases are generally oligomeric proteins consisting of identical subunits. Enzymes from eubacterial and archaeal sources tend to be 19 to 23 kDa subunits and exist as either a tetramer or hexamer. See Alebeek, et al., supra (the disclosure of which is hereby incorporated by reference herein). Dissociation to dimers and trimers has been shown to exist in the absence of divalent cations, Icheba et al., J. Biochem. (1990) 108: 572-578. Eucaryotic enzymes are homodimers of 32 to 35 kDa and are exclusively dimers. The only known exception to these rules is the PPase from Methanothrix soehngenii that posses an .alpha..sub.2 .beta..sub.2 subunit structure, Jetten, et al., supra (the disclosure of which is hereby incorporated by reference herein).
All known PPases require the presence of divalent metal cations, with magnesium conferring the highest activity. One exception to this rule is the PPase from Bacillus subtilis in which the optimal activity occurs in the presence of Mn.sup.2+. See Tono and Kornberg, J. Biol. Chem. (1967) 242:2375-2382, the disclosure of which is hereby incorporated by reference herein. Kinetic studies have shown that the complex of Mg.sup.2+ and PP.sub.i (MgPP.sub.i.sup.2-) is the true substrate and not PP.sub.i alone. The addition of free magnesium ions activates the enzyme by direct binding, while free PP.sub.i is thought to inhibit the enzyme. For details of PPase kinetics, see Rapoport, et al., Eur. J. Biochem. (1972) 26:237-246 and Lahti, R., supra (the disclosures of which are incorporated by reference herein).
One interesting feature of this enzyme is its unusual thermostability; the inorganic pyrophosphatase appears to be one of the most thermostable enzymes of an organism. Research has shown that the thermostability of inorganic pyrophosphatases is increased by the presence of divalent cations. Commercially available PPases are heat resistant in the presence of Mg.sup.2+ up to the following temperatures: Escherichia coli (80.degree. C.), Saccharomyces cerevisiae (Baker's yeast) (50.degree. C.) and Bacillus stearothermophilus (80.degree. C.). Heat resistance is defined as the temperature at which the activity of the enzyme decreases not more than 5% within 10 minutes. See, Schreier, E. and Hohne, W. E., FEBS Letters (1978) 90: 93-96, the disclosure of which is hereby incorporated by reference herein.
The thermostability of other inorganic pyrophosphatases has also been studied. The two thermophilic PPases from thermophilic bacterium PS-3 and Bacillus Stearothermophilus were found to be thermostable to 75.degree. C. in the presence of Mg.sup.2+ ; alone in Tris-HCl buffer these two thermophilic PPases were not intrinsically stable, with the PS-3 PPase losing activity at 40.degree. C. See, Hachimori, et al., J. Biochem. (1978) 86:121-130, the disclosure of which is hereby incorporated by reference herein. The PPase from Sulfolobus acidocaldarius strain 7 retains complete activity after incubation at 100.degree. C. for 10 minutes in the presence of MgCl.sub.2 ; see, Wakagi, et al., supra. The PPase from Thermus thermophilus is heat killed at 96.degree. C. and retains 50% activity after incubation at 90.degree. C. for one hour in the presence of MgCl.sub.2 ; see Kuranova, et al., Dokl. Akad. Nauk. SSSR (1987) 295:1013-1016 and Hohne, et al., Biomed. Biochim. Acta (1988) 47:941-947, the disclosures of which are hereby incorporated by reference herein. The PPase from Thiobacillus thiooxidans retains 90% activity at 80.degree. C. and 40% activity at 100.degree. C. in the presence of Mg.sup.2+ ; see, Tominga, N. and Mori, T.,J. Biochem. (1977) 81:477-483, the disclosure of which is hereby incorporated by reference herein. The PPase from Thiobacillus ferrooxidans retains 8% activity after incubation at 100.degree. C. for 60 minutes; see, Howard, A. and Lundgren, D. G., Can. J. Biochem. (1970) 48:1302-1307, the disclosure of which is hereby incorporated by reference herein.
Research has been conducted on the effect of pyrophosphorolysis (the reverse reaction of polymerization) and the use of pyrophosphatases in DNA sequencing. Tabor and Richardson, J. Biol. Chem. (1990) 265:8322-8328, the disclosure of which is hereby incorporated by reference herein, discovered that pyrophosphorolysis by bacteriophage T7 DNA polymerase can lead to degradation of specific dideoxy-nucleotide-terminated fragments on DNA sequencing gels. The variation in band intensities in a DNA sequencing reaction could be prevented by the addition of inorganic pyrophosphatase to reduce the level of inorganic pyrophosphate. Uniform band intensities are extremely helpful in the analysis of a DNA sequence, particularly with automated DNA sequencers.
Moreover, research has been conducted on the use of inorganic pyrophosphatase to improve the yield of in vitro transcription reactions catalyzed by T7 RNA polymerase. See Cunningham and Ofengand, Biotechniques (1990) 9:713-714, the disclosure of which is hereby incorporated by reference herein.
Accordingly, there is a desire in the art to obtain and produce a purified, highly thermostable inorganic pyrophosphatase that may be used to eliminate the problems of pyrophosphorolysis including reactions higher than 37.degree. C. up to 100.degree. C. or higher, in any such process where an accumulation of pyrophosphate could be a problem; for example, any process where DNA polymerases are used in recombinant DNA technology, such as thermal cycle sequencing and primer extension reactions.